Progressive Multifocal Leukoencephalopathy (PML) is a demyelinating disease of the CNS caused by reactivation of the polyomavirus JC (JCV). PML occurs in immunosuppressed people, including in up to 5% of patients with AIDS. We have identified epitopes of JCV recognized by CD8+ cytotoxic T lymphocytes (CTL) of HLA A2+ patients and shown that despite their low frequency in peripheral blood, these cells are instrumental in improving PML survival. We have demonstrated that autologous dendritic cells (DC) can efficiently present these epitopes to CTL and expand this cellular immune response in vitro. Conversely, the role of CD4+ T cells, which may display a regulatory phenotype, and potentially down modulate this immune response in patients who rapidly succumb to PML, has not been investigated in details. We hypothesize that PML progressors, who die within one year of disease onset, have T lymphocytes displaying a regulatory phenotype or a Th2 cytokine production pattern, as well as markers of T cell exhaustion that down modulate their antiviral activity. Furthermore, we postulate that JCV contains immunodominant CTL epitopes restricted by commonly expressed class I alleles other than A2, which will allow ex vivo detection of JCV immune responses, and that can be used to develop a DC-based immunotherapy to benefit all PML patients. We also postulate that we can improve the potency of JCV-peptide pulsed DC vaccines by further amplifying the T cell response with T cell-specific CD3/CD28 antibodies and by blocking the PD-1 pathway leading to T cell exhaustion. Finally, we hypothesize that transfection of DC with mRNA coding for JCV proteins will elicit a broad and potent stimulation of anti-JCV cellular immune response mediated by both CD4+ and CD8+ T cells in vitro. These studies will pave the way for a treatment trial for PML using DC vaccines. To test these hypotheses, we will pursue the following set of specific aims: Aim 1) Characterize the cytokine expression profile of JCV-specific CD4+ and CD8+ T cells in PML patients with different clinical outcomes Aim 2) Search for CTL epitopes of JCV restricted by common HLA class I alleles and characterize the immune response to these epitopes in fresh blood and cultured cell samples. Aim 3) Maximize the efficiency of JCV peptide-pulsed DC vaccines by sequential stimulation of T cells with anti- CD3/CD28 antibodies and blockade of PD-1 receptor to limit immune exhaustion. Aim 4) Determine the potential of JCV mRNA-transfected DC as therapeutic vaccines for PML